Coinheritance of BRCA1 and BRCA2 mutations with Fanconi anemia and Bloom syndrome mutations in Ashkenazi Jewish population: possible role in risk modification for cancer development.

Fanconi anemia (FA) and Bloom syndrome (BS) are rare autosomal recessive genetic disorders manifesting in childhood, with a predisposition to cancer development in adolescence and adulthood. Both syndromes are relatively prevalent among the Ashkenazi Jewish population, and, in both syndromes, mutations specific to this population have been identified. Similarly, unique Ashkenazi mutations were found in the genes BRCA1 and BRCA2. These two genes, when mutated, play important roles in familial breast and ovarian carcinogenesis. The genes involved in the pathogenesis of the FA and BS belong to the general class of instability genes. Heterozygosity for the FA gene has no known promalignant potential, while the BS mutation carrier state was associated with an increased frequency of colorectal cancer. The especially frequent carrier state among the Ashkenazi Jewish population coupled with the high prevalence of BRCA1 and BRCA2 in the same population has led us to search for coinheritance affecting the potential for cancer development. One hundred Ashkenazi women with known BRCA1 and BRCA2 mutations were screened for the FA mutation IVS4+4 A-->T and the BS mutation blm(Ash). Our results indicate that there is an increased prevalence of both FA and BS mutation carriers among the population studied compared with the general Ashkenazi population (prevalence of FA mutation 4/100 women [4%] as compared to 35/3104 previously published controls [1.1%], P=0.031, and for BS mutation 3/100 [3.2%] as compared to 36/4001 [0.9%], P=0.058). There was no statistically significant effect of the coinheritance on cancer prevalence, type of cancer, or age of cancer onset. Coinheritance of FA and/or BS mutations seems to be more prevalent among BRCA mutation carriers, but a larger study encompassing more women may help in clarifying this issue.

Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (Duet) combined morphological and cytogenetical analysis.

Recurrent disease remains a major obstacle to cure after allogeneic transplantation. Various methods have been developed to detect minimal residual disease (MRD) after transplantation to identify patients at risk for relapse. Chimerism tests differentiate recipient and donor cells and are used to identify MRD when there are no other disease-specific markers. The detection of MRD does not always correlate with relapse risk. Chimerism testing may also identify normal hematopoietic cells or other cells not contributing to relapse. In this study we report our initial experience with a novel system that provides combined morphological and cytogenetical analysis on the same cells. This system allows rapid automatic scanning of a large number of cells, thus increasing the sensitivity of detection of small recipient population. The clinical significance of MRD detection is improved by identifying the morphology of recipient cells. Identification of recipient characteristics within blasts predicts overt relapse in leukemia patients and precedes it by a few weeks to months. Identification within mature hematopoietic cells may not be closely associated with relapse. The system also allows chimerism testing after sex-mismatched transplants, within cellular subsets, with no need for sorting of cells. The system merits further study in larger scale trials.

Activated macrophages for treating skin ulceration: gene expression in human monocytes after hypo-osmotic shock.

Macrophages play a major role in almost all stages of the complex process of wound healing. It has been previously shown that the incorporation of a hypo-osmotic shock step, in the process of monocyte-concentrate preparation from a blood unit, induces monocyte/macrophage activation. As the macrophages are produced using a unique, closed and sterile system, they are suitable for local application on ulcers in elderly and paraplegic patients. Enhanced phagocytosis by the activated cells, as well as increased secretion of cytokines such as IL-1, IL-6, were detected in a recent study which are in accord with the very encouraging clinical results. In the present study, we used DNA microarrays to analyse the differential gene expressions of the hypo-osmotic shock-activated monocytes/macrophages and compare them to non-treated cells. Of the genes that exhibited differences of expression in the activated cell population, 94% (68/72) displayed increased activity. The mRNA levels of 43/68 of these genes (63%) were found to be 1.5-fold or higher (1.5-7.98) in the activated macrophages cell population as compared to the non-treated cells. Only four genes were found to have lower mRNA levels in the activated cells, with ratios of expression of 0.62-0.8, which may suggest that the changes are insignificant. A significant number of the genes that showed increased levels of expression is known to be directly involved in macrophage function and wound healing. This may correlate with the increased secretion of different cytokines by the activated macrophages depicted previously. Other groups of genes expressed are known to be involved in important pathways such as neuronal growth and function, developmental defects and cancer. The hypo-osmotic shock induces a gene expression profile of cytokines and receptors in the activated cells. These may evoke potential abilities to produce a variety of protein products needed in the wound healing process and may bring to light possibilities for other therapeutic applications of these cells.

Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less).

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma.

Plakoglobin and its homologue beta-catenin are cytoplasmic proteins that mediate adhesive functions by interacting with cadherin receptors and signaling activities by interacting with transcription factors. It has been suggested that plakoglobin can suppress tumorigenicity whereas beta-catenin can act as an oncogene. We investigated the correlation between the expression pattern of N-cadherin, beta-catenin, and plakoglobin and tumor behavior in primary tumors of 20 neuroblastoma patients of all stages and in 11 human neuroblastoma cell lines. N-cadherin and beta-catenin were detected in 9 of 11 and 11 of 11 cell lines, respectively, whereas plakoglobin was undetectable or severely reduced in 6 of 11 cell lines. Tumor cells from 16 of 20 patients expressed N-cadherin and 20 of 20 patients expressed beta-catenin at levels similar to those of normal ganglion cells. Plakoglobin was undetectable in 9 of 20 tumors. Plakoglobin deficiency in the primary tumors was significantly associated with adverse clinical outcome. Five of the patients with plakoglobin-negative tumors died whereas four patients are alive without evident disease. In contrast, all patients with plakoglobin-positive tumors are alive; 2 of 11 are alive with the disease and 9 of 11 are alive without evident disease. These results suggest that down-regulation of plakoglobin may be of prognostic value for neuroblastoma patients as predictor of poor outcome.

Subgroup of patients with Philadelphia-positive chronic myelogenous leukemia characterized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis.

A major deletion of the region proximal to the rearranged ABL gene on 9q was found in 14/94 (15%) of chronic myelogenous leukemia Philadelphia-positive patients by interphase fluorescent in situ hybridization with the BCR/ABL extra signal dual-color probe. Preliminary results indicated that the prognosis of the deletion 9q patients is probably worse than that of the non-deletion 9q patients. Twelve of the 14 deletion 9q patients were treated with alpha-interferon and none had a major cytogenetic response. The median duration of the chronic phase in patients not undergoing BMT was significantly shorter for the deletion 9q patients as compared to the non-deletion 9q patients (p =.0144). DNA microarray technology was performed in order to compare the gene expression patterns between the two groups of patients. A number of genes exhibiting differential expression, especially involving cell adhesion and migration, were identified. This finding may identify a sub-group of CML patients with different cell properties and a relatively poor prognosis.

Detection of unidentified chromosome abnormalities in human neuroblastoma by spectral karyotyping (SKY).

Spectral karyotyping (SKY) is a novel technique based on the simultaneous hybridization of 24 fluorescently labeled chromosome painting probes. It provides a valuable addition to the investigation of many tumors that can be difficult to define by conventional banding techniques. One such tumor is neuroblastoma, which is often characterized by poor chromosome morphology and complex karyotypes. Ten primary neuroblastoma tumor samples initially analyzed by G-banding were analyzed by SKY. In 8/10 tumors, we were able to obtain additional cytogenetic information. This included the identification of complex rearrangements and material of previously unknown origin. Structurally rearranged chromosomes can be identified even in highly condensed metaphase chromosomes. Following the SKY results, the G-banding findings were reevaluated, and the combination of the two techniques resulted in a more accurate karyotype. This combination allows identification not only of material gained and lost, but also of breakpoints and chromosomal associations. The use of SKY is therefore a powerful tool in the genetic characterization of neuroblastoma and can contribute to a better understanding of the molecular events associated with this tumor.

Activation of human monocytes/macrophages by hypo-osmotic shock.

Phagocytosis and secretion of interleukins and growth factors put the macrophage in the centre of the wound healing process. For the last four years over 400 human ulcers have been treated in elderly and paraplegic patients by local application of monocytes prepared from a blood unit, in a unique, closed, sterile system. The process of preparation includes a step of hypo-osmotic shock, which induces monocyte/macrophage activation. This is different from any other known method of activation. In the present study we evaluated the efficacy of the hypo-osmotic shock. We found enhanced levels of IL-1 (P = 0.004) and IL-6 (P = 0.001) in the incubation medium (100% autologous serum) of the activated cells, as compared with controls, prepared in the same system. The IL-1 reached a plateau after 6 and 12 h incubation at 37 degrees C, in both experimental and control incubation medium. The level of IL-6 was further elevated after 12 and 24 h incubation in experimental and control incubation mediums (P = 0.001). The phagocytosis of fluorescent beads was markedly enhanced after hypo-osmotic shock (P = 0.005). The osmotic shock induced macrophages were compared to those stimulated with LPS, and osmotic shock was proved to be at least as efficient method of stimulation as LPS.

Engraftment-associated hypophosphatemia--the role of cytokine release and steep leukocyte rise post stem cell transplantation.

Hypophosphatemia associated with bone marrow transplantation has been infrequently reported. The suggested mechanism is phosphate uptake by the replicating cells. Various cytokines are associated with the development of hypophosphatemia. The present study evaluated the interrelationship between cytokine release, the rise in WBC and the development of hypophosphatemia during the engraftment period. Blood samples were obtained from 60 patients undergoing peripheral blood stem cell transplant, on the day of admission and then daily from the day of transplant until discharge. Hypophosphatemia developed in 62% of the patients. The median day of minimal phosphorus level was +8 and it antedated engraftment by 2 days. There was a significant correlation between the day of minimal phosphorus level and the day of maximal WBC and a significant correlation between the fall in phosphorus level and WBC rise. IL-6 and IL-8 showed similar kinetics. Higher IL-6 and IL-8 levels were directly associated with lower phosphorus levels. In conclusion, hypophosphatemia commonly occurs in the post-transplant period. We assume that both a direct effect of cytokine release and an increased consumption by the dividing WBCs contribute to its appearance. As its occurrence usually antedates engraftment it can be used as a forerunner for WBC recovery.

Mutation analysis of the FAS and TNFR apoptotic cascade genes in hematological malignancies.

OBJECTIVE: The existence of properly functioning apoptotic pathways is of utmost importance in the maintenance of a normal cell count. Several groups have searched for mutations in the FAS receptor, a well-characterized apoptotic protein carrying a death domain, and reported the existence of rare mutations in multiple myeloma, T-acute lymphoblastic leukemia (T-ALL), and adult T-cell leukemia. Our aim was to expand these searches by looking for mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP, in the promoter region of FAS, and in the protease domain of caspase 10, in a larger variety of hematological malignancies, some of which express an apoptosis-resistant phenotype. METHODS: We extracted RNA and DNA samples from 92 hematological malignancies: chronic lymphocytic leukemia (CLL; 31 cases), chronic myelogenous leukemia (CML; 28 cases), essential thrombocythemia (ET; 8 cases), acute lymphocytic leukemia (ALL; 6 cases), acute myeloblastic leukemia (AML; 6 cases), hairy-cell leukemia (HCL; 3 cases), Burkitt's lymphoma (3 cases), polycythemia vera (PV; 3 cases), myelofibrosis (2 cases), and chronic myelomonocytic leukemia (CMML; 2 cases) and performed PCR-SSCP and sequence analysis on these samples. RESULTS: Five polymorphic patterns were found: three in the death domain of the FAS gene in CML patients, one in the promoter of this gene in a CLL patient, and the fifth in the death domain of the TRADD gene in a CML patient. No mutations, altering amino acids, were found in these genes in any of the aforementioned malignancies. CONCLUSIONS: These observations imply that mutations in the death domains of FAS, FADD, TNFR, TRADD, and RIP and in the protease domain of caspase 10 are not a major cause for failure of apoptosis in hematological malignancies, mainly CML and CLL. Regulatory and epigenetic abnormalities in these apoptotic cascade members and aberrations in other components of all death machinery should be looked for.

Spontaneous regression of congenital leukaemia with an 8;16 translocation.

Congenital leukaemia (CL) is a rare disorder that presents with extramedullary infiltrates and a myeloid phenotype. CL can progress rapidly without adequate treatment, but, paradoxically, may also remit spontaneously. Because of the significant toxicity involved in delivering chemotherapy to newborns, it is important to identify those newborns who may not require treatment. We describe an infant who presented at 1 week of age with congenital myeloid leukaemia. Cytogenetic analysis revealed a t(8;16)(q11;p13) translocation. The infant's leukaemia underwent a spontaneous regression. This case further confirms the possibility of spontaneous remission in congenital leukaemia. Moreover, it suggests that the presence of a clonal cytogenetic aberration does not preclude the possibility of a spontaneous regression in CL.

Autosomal-dominant giant platelet syndromes: a hint of the same genetic defect as in Fechtner syndrome owing to a similar genetic linkage to chromosome 22q11-13.

Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations-deafness, cataracts, and nephritis-and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.

Amplification of immunological functions by subcutaneous injection of intermediate-high dose interleukin-2 for 2 years after autologous stem cell transplantation in children with stage IV neuroblastoma.

BACKGROUND: Immunotherapy given post-autologous stem cell transplantation may eliminate residual tumor cells escaping the conditioning protocol. METHODS: Five children suffering from stage IV neuroblastoma were treated by recombinant interleukin-2 (IL-2) post-autologous peripheral blood stem cell transplantation. The patients' peripheral mononuclear cells were monitored for CD3+ and CD56+ levels, their proliferative response and killing of various cell lines targets. RESULTS: An increase in the level of total lymphocytes, mainly due to expansion of T cells, and enhanced proliferative response to phytohemaglutinin were observed. Elevated cytotoxicity against K562 and neuroblastoma target cells was detected in four patients and against K562 targets in one patient. Toxicity included mild thrombocytopenia, and fever in four patients and mild to moderate encephalopathy which necessitated withdrawing one patient from the protocol. Three of five patients studied are alive today, one of them whose IL-2 was stopped, is in relapse. Two patients have died. CONCLUSIONS: Immunotherapy with s.c. intermediate-high dose IL-2 is feasible and results in expansion of T cells and in stimulation of killing activity against several targets including in some cases, neuroblastoma tumor cells.

Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH), as a new clinical test, is not presently standardized. For practical reasons, each laboratory must build its own criteria. In this work, we present our standardization criteria for clinical practice, which include not only the methods for cell fixation, specimen preparation, and hybridization conditions, but mainly the definition of false-positive range and the scoring criteria of microscopic analysis. These include signal assessment, difference between individual microscopists, evaluation of specimen homogeneity, and the minimum number of scored nuclei required for a clinically reliable result. For this purpose, we analyzed by FISH 24 healthy volunteer donors, 31 patients affected by non-chronic myelogenous leukemia (CML) hematological malignancies, 47 CML patients at diagnosis, and 82 CML patients during treatment for the BCR/ABL fusion. In this article, we present several quality control and assurance methods that can be useful in providing standardization of the FISH technique.

Genetic linkage of autosomal-dominant Alport syndrome with leukocyte inclusions and macrothrombocytopenia (Fechtner syndrome) to chromosome 22q11-13.

Fechtner syndrome is an autosomal-dominant variant of Alport syndrome, manifested by nephritis, sensorineural hearing loss, cataract formation, macrothrombocytopenia, and polymorphonuclear inclusion bodies. As opposed to autosomal-recessive and X-linked Alport syndromes, which have been genetically well studied, the genetic basis of Fechtner syndrome remains elusive. We have mapped the disease-causing gene to the long arm of chromosome 22 in an extended Israeli family with Fechtner syndrome plus impaired liver functions and hypercholesterolemia in some individuals. Six markers from chromosome 22q yielded a LOD score >3.00. A maximum two-point LOD score of 7.02 was obtained with the marker D22S283 at a recombination fraction of 0. Recombination analysis placed the disease-causing gene in a 5.5-Mb interval between the markers D22S284 and D22S1167. No collagen genes or genes comprising the basement membrane have been mapped to this region.

Plasma glutathione peroxidase deficiency and platelet insensitivity to nitric oxide in children with familial stroke.

In a previous report by Freedman et al (J Clin Invest. 1996;97:979-987), plasma from 2 brothers with stroke or transient ischemic attack inactivated the antiplatelet effects of nitric oxide (NO), and this effect was found to be a consequence of a deficiency of plasma glutathione peroxidase (GSH-Px). In this study, we attempted to define the generalizability of this deficiency by studying NO-mediated antiplatelet effects in 7 families with familial childhood stroke. Seven families with familial childhood stroke that consecutively presented to a large referral center were included in the study. We monitored ADP-induced aggregation of normal gel-filtered platelets (GFP) in platelet-poor plasma (PPP) from normal individuals and from patients in the presence or absence of an NO donor (S-nitroso-glutathione). Surface P-selectin expression of normal GFP in patients' PPP was analyzed by flow cytometry after incubation with a P-selectin-specific monoclonal antibody in the presence or absence of the NO donor. We also measured GSH-Px activity in plasmas from family members and normal controls using standard methods. In 6 of 7 families, NO failed to inhibit platelet P-selectin expression and platelet aggregation in PPP from the affected family members and some of their relatives. Of 4 families studied, 3 probands and their corresponding affected parent had 50% decrease in plasma GSH-Px activity. In some patients with childhood stroke, impaired metabolism of reactive oxygen species as a result of reduced GSH-Px activity results in NO insufficiency that affects normal platelet inhibitory mechanisms and predisposes to arterial thrombosis.

Successful human umbilical cord blood stem cell transplantation without conditioning in severe combined immune deficiency.

A 2-month-old girl with severe combined immunodeficiency (SCID), presented with mild staphylococcal skin infection, lymphopenia, low T cell number, absence of B cells, high number of NK cells, and a negligible response to mitogens. Since her older brother died as a result of SCID 2 years earlier, cord blood was harvested from a sister born 2 1/2 years earlier, who was normal and fully matched both by serology and molecular typing. In view of her clinical condition and in spite of a high number of NK cells with normal activity, HUCBT without preparative conditioning was performed. No G-CSF was administered. Engraftment with mixed chimerism was evident 3 weeks post transplantation. There were no peritransplantation complications. Eighteen months post transplantation, the girl is in excellent condition, blood counts are normal, T cell engraftment is complete, B cell engraftment is proceeding gradually, and the mitogen stimulation tests are normal. Due to the unique nature of HUCB hematopoietic cells, engraftment without conditioning may be possible in patients with SCID with fully matched donors. This is the first HUCBT performed without conditioning.

Coexistence of several unbalanced translocations in a case of neuroblastoma: the contribution of multicolor spectral karyotyping.

Spectral karyotyping (SKY) is based on the simultaneous hybridization of a set of 24 chromosome-specific DNA painting probes, each labeled with a different fluor combination. Automatic classification, based on the measurement of the spectrum for each chromosome, was applied to metaphases obtained from the affected bone marrow of a neuroblastoma case. Spectral karyotyping allowed the identification of chromosomal aberrations that could not be identified by the use of the G-banding technique, and revealed a number of gains and unbalanced translocations.

Fast-FISH detection and semi-automated image analysis of numerical chromosome aberrations in hematological malignancies.

A new fluorescence in situ hybridization (FISH) technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available alpha-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, and microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

Instability of dinucleotide repeats in Hodgkin's disease.

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor suppressor genes. However, the investigation of genomic instability of microsatellites has disclosed a new mechanism for human carcinogenesis, which is involved not only in hereditary nonpolyposis colon cancer (HNPCC) but in a number of other malignancies as well. To determine whether microsatellite instability is involved in Hodgkin's disease, we screened 16 such tumors using 7 microsatellite marker loci on 6 chromosome arms 4, 5, 9p, 9q, 11, 14, and 17. Using the polymerase chain reaction method, DNA samples from the tumors and from normal peripheral blood leukocytes from each patient were compared for the allelic pattern produced at each locus. Five cases of genomic instability were identified, suggesting that this mechanism is relevant to the pathogenesis of HD.